Development of a single-tube duplex EvaGreen real-time PCR for the detection and identification of EHV-1 and EHV-4.
Abstract: The objective of this study was to develop a novel EvaGreen (EG) based real-time PCR technique for the simultaneous detection of Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) genomes from equine nasal swabs. Viral genomes were identified based on their specific melting temperatures (T m), which are 88.0 and 84.4 °C for EHV-1 and EHV-4, respectively. The detection limitation of this method was 50 copies/μl or 0.15 pg/μl for EHV-1 and 5 copies/μl or 2.5 fg/μl for EHV-4. This assay was 50-1,000 times more sensitive than the SYBR Green (SG)-based assay using the same primer pairs and as sensitive as the TaqMan-MGB probe-based assay. The validity of the real-time PCR assays was confirmed by testing 13 clinical samples. When all results of the EG, SG, and TaqMan probe-based singleplex and duplex real-time PCRs were considered together, a total of 84.6 % (11/13) horses and donkeys were positive for at least one virus. EHV-1 and EHV-4 coexisted in 81.8 % (9/11) horses. Overall, we report that the EvaGreen duplex real-time PCR is an economical and alternative diagnostic method for the rapid differentiation of EHV-1 and EHV-4 in nasal swabs.
Publication Date: 2014-03-11 PubMed ID: 24615388DOI: 10.1007/s00253-014-5626-6Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Validation Study
Summary
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The research is about the development and validation of a more economical and effective method based on EvaGreen real-time PCR for simultaneous detection and differentiation of EHV-1 and EHV-4 from horse nasal samples.
Experiment Overview
- The goal of the research was to create a new PCR technology known as EvaGreen (EG) real-time PCR for detecting Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) from equine nasal swabs.
- The researchers distinguished the viral genomes based on their melting temperatures where EHV-1 and EHV-4 had melting temperatures of 88.0 and 84.4 °C respectively.
- The research compared the efficiency and sensitivity of this technique with other PCR techniques such as SYBR Green (SG)-based assay and TaqMan-MGB probe-based assay.
Results and Observations
- The limitations for detecting EHV-1 and EHV-4 with the EvaGreen real-time PCR were found to be 50 copies/μl or 0.15 pg/μl and 5 copies/μl or 2.5 fg/μl, respectively. It means that the technique was capable of detecting minute amounts of viral genome in the samples.
- The EvaGreen-based assay proved to be between 50 and 1,000 times more sensitive than the SYBR Green-based assay, using the same primer pairs, and was observed to have a comparable sensitivity to the TaqMan-MGB probe-based assay, which is considered accurate.
- The applicability and accuracy of the EvaGreen real-time PCR were also validated by testing 13 clinical samples. It was found that 84.6 % of the animals were carrying at least one of the viruses, and coexistence of EHV-1 and EHV-4 was observed in 81.8% of the horse samples.
Conclusion
- Based on the results, the researchers concluded that the EvaGreen duplex real-time PCR is a highly efficient and cost-effective method for the early detection and differentiation of EHV-1 and EHV-4 in nasal swabs.
- This method could serve as a crucial diagnostic tool in return, helping to control and manage the spread of EHV-1 and EHV-4 among horse populations.
Cite This Article
APA
Hu Z, Zhu C, Chang H, Guo W, Liu D, Xiang W, Wang X.
(2014).
Development of a single-tube duplex EvaGreen real-time PCR for the detection and identification of EHV-1 and EHV-4.
Appl Microbiol Biotechnol, 98(9), 4179-4186.
https://doi.org/10.1007/s00253-014-5626-6 Publication
Researcher Affiliations
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, 427 Maduan Street, Harbin, 150001, People's Republic of China.
MeSH Terms
- Animals
- DNA, Viral / chemistry
- DNA, Viral / genetics
- Equidae
- Herpesviridae Infections / diagnosis
- Herpesviridae Infections / veterinary
- Herpesviridae Infections / virology
- Herpesvirus 1, Equid / isolation & purification
- Herpesvirus 4, Equid / isolation & purification
- Horses
- Molecular Diagnostic Techniques / methods
- Nasal Mucosa / virology
- Real-Time Polymerase Chain Reaction / methods
- Sensitivity and Specificity
- Staining and Labeling / methods
- Temperature
- Transition Temperature
- Veterinary Medicine / methods
Citations
This article has been cited 1 times.- Nielsen SS, Alvarez J, Bicout DJ, Calistri P, Canali E, Drewe JA, Garin-Bastuji B, Gonzales Rojas JL, Gortázar C, Herskin M, Michel V, Miranda Chueca MÁ, Roberts HC, Padalino B, Pasquali P, Spoolder H, Ståhl K, Calvo AV, Viltrop A, Winckler C, Carvelli A, Paillot R, Broglia A, Kohnle L, Baldinelli F, Van der Stede Y. Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): infection with Equine Herpesvirus-1. EFSA J 2022 Jan;20(1):e07036.
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